An ion channel is a protein, penetrating a biological membrane, whose basic structure is a lipid bilayer membrane, and adjusts entrance and exit of ions according to stimulus so as to generate an electric signal or a calcium signal in a cell. That is, the ion channel is an important protein molecule for converting stimulus into an intracellular signal.
Such an ion channel is constituted of a pore serving as an ion path and a gate for controlling opening/closing of a channel. This opening/closing function can be confirmed by measuring an ion current at the time when the ion passes through the ion channel. As a method for measuring an ion current of a single ion channel, a patch-clamp method is adopted. Also a planar lipid bilayer method is adopted so as to measure the ion current.
In order to more deeply study correlation of structural functions of the ion channel, it is necessary to use a simple rearrangement system in carrying out an experiment. An artificial lipid bilayer membrane formation method adopted in this case is the planar lipid bilayer method. In the planar lipid bilayer method, a minimum simple system including ion, water, an artificial lipid bilayer membrane, and an ion channel is used so as to study a basic structure of the ion channel and detail correlation of structural functions thereof (Non-Patent Document 1 and the like).
The following specifically explains a system of the planar lipid bilayer method. As illustrated in FIG. 7, an ion channel 112 is provided in an artificial lipid bilayer membrane 111, and a current flowing via the ion channel 112 is measured. The artificial lipid bilayer membrane 111 is formed on a small hole 115 provided in a partition plate 114 such as a plastic plate for parting an aqueous solution chamber 113. In one of two chambers obtained by parting the aqueous solution chamber 113, an electrode 116 is provided. Via the electrode 116, a current measuring device 117 is provided. In the other chamber, an electrode 118 is provided. Via the electrode 118, an earth 119 causes the aqueous solution chamber 113 to be earthed.
Here, examples of how to form the artificial lipid bilayer membrane 111 on the small hole 115 include (A) vertical painting method, (B) vertical applying method, (C) horizontal formation method, and the like.
In the (A) vertical painting method, first, as illustrated in the left illustration of FIG. 8, with a thin glass tube or the like, lipid solution 110 is applied to the small hole 115 provided in a support such as the plate 114 for parting the aqueous solution chamber 113 (not shown in FIG. 8). Under this condition, the lipid solution 110 swells in directions of both surfaces of the partition plate 114 so as to cover the small hole 115. The lipid solution 110 is obtained by dissolving lipid in organic solvent such as decane. After applying the lipid solution 110, the lipid solution 110 moves on the surface of the plate 114 as illustrated in the right illustration of FIG. 8, thereby obtaining an artificial lipid bilayer membrane which has become thinner in a natural manner. Note that, the wording “become thinner” means a process in which the organic solvent or the like moves from a central portion of the applied lipid solution 110 so that a lipid bilayer membrane is formed in the central portion.
Next, in the (B) vertical applying method, as illustrated in the upper illustration of FIG. 9, a lipid monomolecular membrane 121 is developed on a gas-liquid interface of the aqueous solution chamber 113 (not shown in FIG. 9). At this time, a position of the gas-liquid interface is the same as a position of a lower side end of the small hole 115 provided in the partition plate 114. Thereafter, as illustrated in the middle illustration of FIG. 9, a liquid surface (gas-liquid interface) of one chamber (right side of the middle illustration) of two chambers obtained by parting the aqueous solution chamber 113 is raised, thereby developing the monomolecular membrane 121 on the surface of the partition plate 114. On this account, one opening side of the small hole 115 is covered by the monomolecular membrane 121. Thereafter, as illustrated in the lower illustration of FIG. 9, a liquid surface (gas-liquid interface) of the other chamber (left side of the lower illustration) of two chambers obtained by parting the aqueous solution chamber 113 is raised, thereby developing the monomolecular membranes 121 on the surface of the partition plate 114. On this account, also the other opening side of the small hole 115 is covered by the monomolecular membrane 121. As a result, on each opening side of the small hole 115, the monomolecular membrane 121 is applied, so that the artificial lipid bilayer membrane 111 is finally formed.
Next, in the (C) horizontal formation method, the aqueous solution chamber 113 illustrated in FIG. 13 is vertically parted with the partition plate 114. At this time, as illustrated in FIG. 10(a), the small hole 115 provided in the partition plate 114 is covered by the lipid solution 110, and the lipid solution 110 is left until the lipid solution 110 becomes thinner in a natural manner as the artificial lipid bilayer membrane 111. Alternatively, as illustrated in FIG. 10(b), a hydraulic pressure above the small hole 115 is raised in the chamber so that the lipid solution 110 swells downward so as to be thinner, thereby forming the artificial lipid bilayer membrane 111.
However, in any one of the artificial lipid bilayer membrane formation methods, it is difficult to quickly form a stable artificial lipid bilayer membrane 111. That is, in the (A) vertical painting method, it takes several minutes to dozens minutes for the lipid solution 110 to move on the surface of the partition plate 114 and become sufficiently thinner as the artificial lipid bilayer membrane 111. Further, in the (B) vertical applying method, it is essential to carry out a pre-treatment with respect to the small hole 115 with organic solvent such as squalene before forming the artificial lipid bilayer membrane 111, so that such a larger number of steps results in a more complicate formation method. Further, it is general that the artificial lipid bilayer membrane 111 is not formed unless the liquid surface is raised and lowered several times.
Further, in the (C) horizontal formation method, in case of leaving the lipid solution 110 covering the small hole 115 until the lipid solution 110 becomes thinner in a natural manner (in case of FIG. 10(a)), it is impossible to intentionally control the formation of the artificial lipid bilayer membrane. Therefore, it sometimes takes several hours for the lipid solution 110 to become thinner. Further, in case of raising the hydraulic pressure above the small hole 115 in the chamber so that the lipid solution 110 becomes thinner, the obtained artificial lipid bilayer membrane 111 has a thin portion serving as the “lipid bilayer membrane” and a thick portion referred to as a cyclic bulk phase surrounding the thin portion. Thus, the artificial lipid bilayer membrane 111 obtained in this method is based on physicochemical balance of the foregoing portions. Thus, if these portions are physicochemically unbalanced by vibration caused by aqueous solution flow or the like, the artificial lipid bilayer membrane 111 is easily broken. Moreover, it is difficult to exactly control a pressure difference between the upper and lower chambers of the aqueous solution chamber 113, so that the obtained artificial lipid bilayer membrane 111 is likely to be unstable.
In case of adopting the planar lipid bilayer method, it is necessary to realize a great object: to form a stable and durable artificial lipid bilayer membrane.
Incidentally, it is considered that permeation of ions and structural change of ion channel molecules occur at the same time upon opening/closing a gate of the ion channel. In order to clarify a relationship between a structure and a function of the ion channel molecules, it is necessary to use a measuring device which can simultaneously measure the structure and the function of the ion channel molecules.
The inventors of the present invention proposed a current measuring device which improves the foregoing problems of the conventional artificial lipid bilayer membrane and can simultaneously measure the structure and the function of the ion channel molecules (for example, Non-Patent Document 2 and the like). As illustrated in FIG. 11, the current measuring device includes two solution chambers: an upper solution chamber 101 and a lower solution chamber 102. On a central portion of a bottom of the upper solution chamber 101, a film 103 having a small hole 105 is applied. Further, the lower solution chamber 102 has an opening 104 in its bottom, and a cover glass 106 is fixed on the opening 104 with an adhesive. On the cover glass 106, an agarose gel layer (not shown) is formed. Note that, as in the system of the planar lipid bilayer method, an electrode 116 is placed in the upper solution chamber 101, and a current measuring instrument 117 is provided via the electrode 116. In the lower solution chamber 102, an electrode 118 is placed, and an earth 119 causes the lower solution chamber 102 to be earthed via the electrode 1 18.
In the current measuring device, first, a lower portion of the upper solution chamber 101 is moved in the lipid solution so as to form a thick membrane made of lipid solution in the small hole 105. Thereafter, the upper solution chamber 101 is placed in the lower solution chamber 102, and the upper solution chamber 101 is lowered so that the thick membrane formed in the small hole 105 comes into contact with the agarose gel layer formed on the cover glass 106. Here, the pressure (hydraulic pressure) in the upper solution chamber 101 is raised so as to make a thick membrane thinner, thereby forming an artificial lipid bilayer membrane.
In the current measuring device, the pressure in the upper solution chamber 101 is raised, so that it takes less time to form an artificial lipid bilayer membrane (to make the thin membrane thinner). The thus formed artificial lipid bilayer membrane is supported by the agarose gel layer. Thus, even when a pressure is exerted by the upper solution chamber 101, the artificial lipid bilayer membrane is stabilized in upward and downward directions. Further, when the agarose gel layer is made thinner, it is possible to observe the artificial lipid bilayer membrane through a lens 107 whose numerical aperture (NA) is large. Thus, even in case where the ion channel is included in the artificial lipid bilayer membrane, the actual ion channel can be observed. On this account, it is possible to simultaneously measure a channel current and a structure of the ion channel.
Non-Patent Document 1
Bayley, H., Cremer, P. Stochastic sensors inspired by biology, Nature 413, 226-230 (2001)
Non-Patent Document 2
Ide, T., Takeuchi, U., Yanagida, T. Development of an Experimental Apparatus for Simultaneous Observation of Optical and Electrical Signals from Single Ion Channels, Single Mol. 3(2002)1, pages 33-42
However, the conventional current measuring device is insufficient in terms of the stability and the size reduction of the artificial lipid bilayer membrane, so that a current measuring device having higher performance is required.
Specifically, first, the conventional current measuring device is arranged so that: as illustrated in FIG. 12, the artificial lipid bilayer membrane 111 formed on the small hole 105 provided in the film 103 is supported by the agarose gel layer on the cover glass 103, so that the artificial lipid bilayer membrane 111 is stabilized in upward and downward directions. However, both the upper solution chamber 101 and the lower solution chamber 102 are open, a higher pressure in the upper solution chamber 101 causes vibration of aqueous solution flow to destabilize the artificial lipid bilayer membrane 111 in a direction (H direction in FIG. 12) parallel to a bottom of the upper solution chamber 101 (not shown in FIG. 12).
Thus, in the conventional current measuring device, it is impossible to strictly keep a curvature of the artificial lipid bilayer membrane 111. Thus, in the aforementioned (C) horizontal formation method, as in the case of raising the hydraulic pressure above the small hole 115 in the chamber so as to make the membrane thinner (FIG. 10(b)), the artificial lipid bilayer membrane 111 and the cyclic bulk phase are physicochemically unbalanced, so that the artificial lipid bilayer membrane 111 is broken.
Further, in the conventional current measuring device, two solution chambers are used, so that it is difficult to reduce the size of the current measuring device. Thus, it is actually impossible to produce the artificial lipid bilayer membrane on the small-size chip.